Sequencing the chloroplast genome of a jujube genotype (Ziziphus jujuba Mill. 'Fengmiguan') uncovered a 101 bp insertion in the large-single copy region

Abstract Ziziphus jujuba Mill., commonly referred to as jujube, is a species of fruiting buckthorn (family Rhamnaceae) that is frequently found across the Shaanxi, Shanxi, and Hebei provinces of China. The ‘Fengmiguan’ variety of jujube, also known as ‘Honey jar,’ is distinguished by its high yield and sugar content, as well as its strong ability to adapt to different environments. In this study, we sequenced and assembled the chloroplast genome (i.e. the plastome) of ‘Fengmiguan’ jujube using a paired-end short-read sequencing technique. The plastome exhibits a quadripartite structure with a total length of 161,818 bp that consists of a large single-copy region (89,427 bp), a small single-copy region (19,361 bp), and two inverted repeats (26,515 bp). The GC content of the plastome is 36.75%. Annotation of the ‘Fengmiguan’ jujube plastome revealed 123 genes, including 79 protein-coding genes, 36 transfer RNA genes, and eight ribosomal RNA genes. Phylogenetic analysis revealed that the ‘Fengmiguan’ variety is closely related to the ‘Bokjo’ variety. Furthermore, we found four variations between these two varieties of jujube, one of which was a 101 bp insertion. Our findings enhance the current understanding of the phylogenetic relationship between different varieties of Z. jujuba Mill., which could possibly aid in the improvement of genetic breeding and population selection in jujubes.


Introduction
Jujube (Ziziphus jujuba Mill.) is a species of fruiting buckthorn (family Rhamnaceae) that grows well in cold, temperate, and warm climates, and is one of the most important species of the Rhamnaceae family due to its economic, ecological, and social importance (Liu et al. 2020). Jujube has a long history of cultivation in China and is a popular food product due to its high levels of nutrients, including vitamin C and organic acids (Shan et al. 2019). The 'Fengmiguan' variety of jujube, also known as 'Honey jar,' is characterized by its small fruit and high sugar content and is thus ideal for fresh consumption or dehydration (Yao and Guldan 2012). In recent years, the chloroplast genomes of several jujube varieties have been sequenced and subjected to phylogenetic analyses (Zhang et al. 2021). However, the chloroplast genome of the 'Fengmiguan' variety has not yet been reported; therefore, it is necessary to decode the chloroplast genome of this variety and compare it to that of other jujube varieties or related species. In this study, we sequenced the chloroplast genome of 'Fengmiguan' jujube for the first time and determined its phylogenetic position within the Rhamnaceae family; this will provide important information about the 'Fengmiguan' jujube plastome that will be valuable for genetic breeding and population selection.

DNA isolation
A modified cetyltrimethylammonium bromide method (Clarke 2009) was used to extract whole genomic DNA from the leaf samples. DNA purity was assessed using a NanoDrop One UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and DNA integrity was verified by agarose gel electrophoresis.

Library preparation, sequencing, and annotation
The isolated DNA was used for paired-end library construction with an insert size of 200-400 bp, using the official standard protocol, and sequenced on the MGISEQ-2000 platform (BGI, Shenzhen, Guangdong, China). The clean paired-end reads were then assembled using GetOrganelle v1.7.6.1 software (Jin et al. 2020) with the Z. jujuba (KU351660) genome serving as a reference. We annotated the resulting chloroplast genome using CPGAVAS2 software ) and manually corrected it using Geneious Prime software (v2021.1.1).
To determine the phylogenetic position of 'Fengmiguan' jujube within the Rhamnaceae family, we performed phylogenetic analysis of the chloroplast genomes from two outgroups (Elaeagnus conferta and Elaeagnus umbellata) and one Berchemiella, two Ventilago, four Rhamnus, three Berchemia, four Hovenia, four Spyridium, and 11 Ziziphus species (Table 1; Figure 3). Phylogenetic analyses revealed that the 'Fengmiguan' variety is closely related to the 'Bokjo' variety and distantly related to the Ziziphus mauritiana and Ziziphus spina-christi varieties. Upon comparing chloroplast genomes of the 'Fengmiguan' and 'Bokjo' genotypes, we discovered four variations within the intergenic region of the LSC region, including two deletions ('T' and 'A' at positions 34,560 and 54,160, respectively) and two     N N N N N N N N N N N N  RN RN N N N N N 1 1 16 6  161 161 161 161 161 161 6 6 61 6 61 1 1  161 61 61 1 1 1 1  161 1 16 6 61 1 1 1  1 16 161 1 1 16 6  1 1 1 1 1 1 1 1 1 ,81  The dispersed repeats consist of direct (D) and palindromic (P) repeats, connected with red and green arcs. The second track shows the long tandem repeats as short blue bars. The third track shows the short tandem repeats or microsatellite sequences as short bars with different colors. The colors, type of repeat they represent, and the description of the repeat types are as follows: black: c (complex repeat); green: p1 (repeat unit size ¼ 1); yellow: p2 (repeat unit size ¼ 2); purple: p3 (repeat unit size ¼ 3); blue: p4 (repeat unit size ¼ 4); orange: p5 (repeat unit size ¼ 5); red: p6 (repeat unit size ¼ 6). The small single-copy (SSC), inverted repeat (IRa and IRb), and large single-copy (LSC) regions are shown on the fourth track. The GC content along the genome is plotted on the fifth track. Genes are color-coded according to their functional classification. The transcription directions for the inner and outer genes are clockwise and anticlockwise, respectively. The key for the functional classification of the genes is shown in the bottom left corner.

Discussion and conclusions
In this study, we sequenced, assembled, and annotated the chloroplast genome of one genotype from the Z. jujuba Mill. 'Fengmiguan' variety. This variety of jujube is famous for its sweet flavor and crisp texture, as well as its abundant fruit production. The chloroplast genome is a suitable model for phylogenetic investigations, due to its specificity (Hu et al. 2022). The comparison of 'Fengmiguan' and 'Bokjo' plastomes revealed four variations that were present in the 'Fengmiguan' plastome, including two deletions and two insertions. These findings contribute to our understanding of the phylogenetic relationship between different Z. jujuba Mill. varieties, which will facilitate improved genetic breeding and population selection in jujubes.

Author contributions
MY and ML conceived the study. SZ and BL collected the samples and performed the experiments. MY, SZ, BL, and LH analyzed the data and prepared the manuscript. MY and ML revised the manuscript. All authors have reviewed and approved the manuscript.

Ethical approval
Research on the chloroplast genome of plants does not require ethical permission or approval. Figure 3. The phylogenetic tree including two outgroups (Elaeagnus conferta and Elaeagnus umbellata) and one Berchemiella, two Ventilago, four Rhamnus, three Berchemia, four Hovenia, four Spyridium, and 11 Ziziphus species. This tree is based on DNA sequences and was constructed using the maximum-likelihood (ML) method. The numbers next to each node indicate the percentage of bootstrap support for 1000 replicates, and all nodes without numbers are 100% supported by the bootstrap sampling test.

Disclosure statement
No potential conflict of interest was reported by any of the authors.